Chitin oligomers for use in promoting non-leguminous plant growth and development

ABSTRACT

Methods for stimulating the growth of non-leguminous plants are disclosed. In the methods, a non-leguminous plant, a part thereof, or a seedling or seed thereof is contacted with a composition comprising a chitooligosaccharide (CO) having the formula: 
     
       
         
         
             
             
         
       
     
     R1 is —H, —CH 3 , —COCH 3 , —SO 3 H, —SO 3 Na, arabinose, methylated arabinose, fucose, or methylated fucose; R2 is —H, —CH 3 , —COCH 3 , —SO 3 H, —SO 3 Na, arabinose, methylated arabinose, fucose, or methylated fucose; each R3 is independently —H or —COCH 3 ; and n is 0, 1, 2, 3, 4, 5 or 6. As non-limiting examples, the method can be used to stimulate production and yield in a cereal grain crop plant, such as rice, wheat or corn (maize).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/180,953 filed on Jun. 17, 2015, which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under IOS1256664 awardedby the National Science Foundation. The government has certain rights inthe invention.

BACKGROUND

This application relates to chitooligosaccharides that lack a lipidmoiety, and to methods of using such chitooligosaccharides to promotegrowth and/or development of non-leguminous plants.

Plants associate with a wide range of microorganisms that facilitate theacquisition of nutrients and protect them against biotic and abioticstresses. For example, interactions with arbuscular mycorrhizal (AM)fungi are widespread in land plants, and this association aids in theuptake of nutrients from the soil (Harrison, M. J. (2005), Annu. Rev.Microbiol. 59: 19-42). Because AM fungi are obligate symbionts, littleis known about the molecular and genetic basis of this symbiosis.

Much of what is known about AM fungi/plant symbiosis has come fromstudies of the symbiotic association between plants and nitrogen-fixingrhizobium bacteria, which most notably results in the formation ofnitrogen-fixing nodules. Unlike the association between AM fungi andplants, the rhizobium bacteria symbiosis is restricted to specificgroups of plants, primarily legumes (Soltis, D. E., et al. (1995), Proc.Natl. Acad. Sci. USA 92: 2647-2651). However, both interactions aresimilar in that they require chemical communication facilitated by theproduction of diffusible signals by the symbiont (Oldroyd, G. E. (2013),Nat. Rev. Microbiol. 11: 252-263).

Rhizobial bacteria signal to legumes with Nod factors, which arelipochitooligosaccharides (LCOs) containing a chitin backbonesubstituted with a lipid moiety, an N-acyl group, and a number ofadditional groups that vary between Nod factors produced by differentspecies of rhizobia (Dénarié, J. et al. (1996), Annu. Rev. Biochem. 65:503-535). Nod factor perception utilizes a signalling pathway that isalso involved in the establishment of mycorrhizal associations (Oldroyd,G. E. (2013)).

AM fungi also produce diffusible signals that are recognized by the hostplant via the common symbiosis signalling pathway. Research suggeststhat at least two different mycorrhizal signals are active on Medicagotruncatula (Chabaud, M., et al. (2011), New Phytol. 189: 347-355).Similarly, work in rice (Oryza sativa) demonstrates mycorrhizalsignalling that is both dependent and independent of the commonsymbiosis signalling pathway (Gutjahr, C., et al. (2008), Plant Cell 20:2989-3005).

The AM fungus Rhizophagus irregularis produces LCOs (Maillet, F., et al.(2011), Nature 469: 58-63), some of which are sulfated, resulting in astructure very similar to the Nod factor produced by Sinorhizobiummeliloti, the symbiont of M. truncatula. These Myc-LCOs activateresponses in M. truncatula similar to those activated by Nod factor,including the promotion of lateral root outgrowth. Consistent with thesefindings, U.S. Patent Publication No. 2011/0301032 discloses a method ofstimulating a plant by contacting the plant with Myc-LCOs and variantsthereof, wherein the variants all retain a lipid moiety.

The lipid moieties incorporated into the LCOs disclosed by, e.g., U.S.Patent Publication No. 2011/0301032, make it difficult and/or expensiveto synthesize large quantities of such compounds from commonly availablechitooligosaccharide source materials. Furthermore, the lipid moietiesdecrease the solubility of such compounds in water, increasing thechallenges associated with dissolving the compounds in aqueous solutionsfor scaled up application to seeds, seedlings, or plants. Accordingly,there is a need in the art for alternate compositions and methods forstimulating plant growth and/or development that do not have thesedisadvantages in large-scale applications.

BRIEF SUMMARY

In addition to LCOs, AM fungi produce short-chain chitooligosaccharideslacking a lipid moiety (COs), such as CO4 and CO5, that may be involvedin AM/plant interactions (Genre, A., et al. (2013), New Phytol. 198:190-202). Furthermore, longer chain COs, such as CO8, are known tofunction as pathogenic signals that stimulate plant defenses. Theinventors disclose herein that both shorter and longer chain COs can beused to promote the growth and/or development of non-leguminous plants,including, without limitation, of cereal grains, such as rice, wheat orcorn (maize).

The term “non-leguminous plant” refers to plant species that are notclassified as legumes. It is well-known in the art as to which plantspecies are legumes. The term “cereal grain” refers to a grass that iscultivated as a crop for the edible components of its grain (a type offruit known in the art as a caryopsis).

Accordingly, in a first aspect, this disclosure encompasses a method forstimulating the growth of a non-leguminous plant. The method includesthe step of contacting a non-leguminous plant, a part thereof, or aseedling or seed thereof with a composition that includes achitooligosaccharide (CO) having the formula:

where n is 0, 1, 2, 3, 4, 5, or 6; R1 is —H, —CH₃, —COCH₃, —SO₃H,—SO₃Na, arabinose, methylated arabinose, fucose, or methylated fucose;R2 is —H, —CH₃, —COCH₃, —SO₃H, —SO₃Na, arabinose, methylated arabinose,fucose, or methylated fucose; and each R3 is independently —H or —COCH₃.As a result of practicing the method, the growth of the plant isstimulated.

“Stimulated” plant growth means that the quantity, weight and/or size ofone or more parts of the plant is increased, relative to a plant wherethe seed, seedling, plant, or plant part has not been contacted with thecomposition that includes the CO. Such increased quantity, size or massmay include, but is not limited to, increased length of the root system,increased number of crown roots, increased number of lateral roots,increased dry weight, increased shoot length, or some combination ofthese. Such plant growth stimulation can have some beneficial effects onthe plant, including, without limitation, enhancing soil nutrientacquisition, facilitating the establishment of young plants in thefield, and increasing crop plant yield.

In some embodiments, the composition is contacted with one or more leafand/or root surfaces of the non-leguminous plant. In some suchembodiments, the composition further comprises a surfactant. A“surfactant,” also known as a “wetting agent,” is a substance that iscapable of reducing the surface tension of a liquid composition.

In some embodiments, the composition is contacted with a seedling,seedling part or seed of the non-leguminous plant. In some suchembodiments, the seedling, seedling part or seed of the non-leguminousplant is submerged in and subsequently removed from the composition. Insome embodiments, the seedling part may include plant foliage or plantroots.

In some embodiments, the composition is contacted with the plant, plantpart, seedling or seed for about 1 hour to about 96 hours. In some suchembodiments, the composition is contacted with the plant, plant part,seedling or seed for about 6 hours to about 48 hours.

In some embodiments, the concentration of the CO in the composition iswithin the range of about 10⁻³ M to about 10⁻¹⁰ M. In some suchembodiments, the concentration of the CO in the composition is withinthe range of about 10⁻³M to about 10⁻⁹M. In some such embodiments, theconcentration of the CO in the composition is within the range of about10⁻³ M to about 10⁻⁸M.

In some embodiments, the composition further includes water and alcohol.The alcohol acts to increase the solubility of the CO in the aqueouscomposition. In some such embodiments, the alcohol is ethanol.

In some embodiments, the non-leguminous plant is a monocotyledon. Insome such embodiments, the monocotyledon is a cereal grain. Non-limitingexamples of cereal grains that can be used with the method include rice,wheat and corn (maize).

In some embodiments, R1 is —H, R2 is —H, and each R3 is —COCH₃. In somesuch embodiments, n is 2 (the compound is tetra-N-acetylchitotetraose,CO4) or 6 (the compound is octa-N-acetyl-chitooctaose, CO8).

Other features of the disclosed methods will become apparent from areview of the specification, claims, and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows calcium spiking in rice in response to Myc-LCOs, Nodfactors, and CO4. Representative calcium traces from rice atrichoblastson lateral roots treated with 10⁻⁸M Myc-LCOs and LCO isolations fromRhizobium sp. NGR234 and R. tropici, as well as 10⁻⁵ M and 10⁻⁸Mtreatments of CO4. The number of cells showing calcium spiking, relativeto the total number of cells analyzed is indicated.

FIG. 2 shows calcium spiking in rice in response to CO4 and CO8.Representative calcium traces from rice atrichoblasts on lateral rootstreated with 10⁻⁸M and 10⁻⁵ M CO4 and CO8. Plants appear to respondequally well to CO4 as to CO8.

FIGS. 3A, 3B, and 3C show promotion of root system development in riceby NS-LCO, S-LCO, CO4, and CO8. The mean number of lateral roots (FIG.3A), the length of the root system (FIG. 3B) and the number of crownroots (FIG. 3C) produced per rice plant is shown in response totreatments of 10⁻⁸M COs or LCOs. Plants were treated for 24 hours andthen grown for two weeks before assessment. Results displayed are basedon at least two replicated experiments (n≧28). The p-value wascalculated using a t-test, assuming a normal distribution of the data,or a Wilcoxon signed-rank test when a normal distribution was notobserved. Significance was determined within a 95% confidence interval.Error bars indicate standard error.

FIGS. 4A, 4B, and 4C show that lateral root induction is dependent uponthe symbiotic signalling pathway. The mean percentage of lateral roots(FIG. 4A), the length of the root system (FIG. 4B) and the number ofcrown roots (FIG. 4C) produced per rice plant is shown in response totreatments of 10⁻⁸M COs or LCOs in wild-type rice plants as well asOspollux and Osdmi3 mutants. Plants were treated for 24 hours and thengrown for two weeks before assessment. Results displayed are based on atleast two replicated experiments and calculated as percentage overaverage of the control treatment (n≧28). The p-value was calculatedusing a t-test, assuming a normal distribution of the data, or aWilcoxon signed-rank test when a normal distribution was not observed.Significance was determined within a 95% confidence interval. Error barsindicate standard error.

FIG. 5 shows induction of calcium spiking in rice trichoblasts andatrichoblasts. The graph shows the percentage of calcium responsivecells among trichoblasts and atrichoblasts of rice. Treatments of 10⁻⁵ Mand 10⁻⁸ M CO4 and the response of trichoblasts near R. irregularishyphae were analyzed.

FIG. 6 shows calcium responses to LCOs and COs in rice trichoblasts withrepresentative calcium traces of root hair cells (trichoblasts) treatedwith mixes of 10⁻⁸ M CO4, S-LCO, and NS-LCO. Note that mixes of Myc-LCOswith CO4 induced calcium oscillations, but the Myc-LCOs alone did not.The number of cells showing calcium responses, relative to the totalnumber of cells analyzed is indicated.

FIG. 7 shows promotion of lateral root emergence in M. truncatula byNS-LCO, but not by CO4. M. truncatula roots were treated with 10⁻⁷ M CO4or 10⁻⁷ M NS-LCO and the effect on lateral root emergence was measured.The numbers in parentheses indicate the number of plants analyzed. Thesignificance of the difference to the treated control plants, asmeasured using a t-test, is indicated. The treatments are measured asfold induction relative to the control. Error bars represent thestandard error.

FIG. 8 shows the in vitro effect of seed application on HRSW root systemlength. Compared with control treatment, CO4 treatment significantlypromoted root growth. One star (*) indicates significant difference atthe P<0.05 (n=15).

FIG. 9 shows the “in pot” effect of seed application on HRSW seedlinggrowth. Compared with control treatment, CO4 and CO8 treatmentsignificantly promoted plant growth. One star (*) indicates significantdifference at the P<0.05 (n=20).

FIG. 10 shows the effect of seed application on rice shoot and rootlength. Rice seeds were treated with three different solutions of 0.5%ethanol (control), 10⁻⁶ M CO4, or 10⁻⁶ M CO8, and grown on modifiedFahraeus medium for 33 days. The length of roots (left bar) and shoots(right bar) were measured. Compared with ethanol treatment, CO4treatment significantly promoted shoot and root growth. One star (*) andtwo stars (**) indicate significant difference at the P<0.1, P<0.01level each. (n=10).

FIG. 11 shows the effect of seed application on corn lateral rootdevelopment. Compared with control treatment, CO4 treatmentsignificantly promoted lateral root production. One star (*) indicatessignificant difference at the p<0.1 (n=20).

FIGS. 12 A and 12B show the effect of foliar application on the shootand root length. Three-weeks-old rice leaves were treated with threedifferent solutions of 0.5% ethanol, 10⁻⁶ M CO4, or 10⁻⁶M CO8 bypainting brushes, and the length of shoots (12A) and roots (12B) weremeasured at treatment day (12A left bar), 14 days after treatment (12Acenter bar), and 18 days after treatment (12A right bar and 12B). Bothshoot and root lengths of CO4 treated rice were significantly longerthan ethanol treated ones at 14 days and 18 days. One star (*) and twostars (**) indicate significant difference at the P<0.1, P<0.01 leveleach (n=11).

DETAILED DESCRIPTION A. In General

This invention is not limited to the particular methodology, protocols,or reagents described, as these may vary. The terminology used herein isfor the purpose of describing particular embodiments only and is notintended to limit the scope of the present invention, which will belimited only by the appended claims.

As used herein and in the appended claims, the singular forms “a”, “an”and “the” include plural reference, unless the context clearly dictatesotherwise. As well, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. Also, the terms “comprising”,“including” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, certain exemplarymethods and materials are now described.

B. The Invention

The present invention provides methods and compositions for promotingplant growth and/or development in non-leguminous plants.

The method includes the step of contacting a non-leguminous plant, apart thereof, or a seedling or seed thereof with a composition thatincludes a chitooligosaccharide (CO), as described above.

The method used for contacting the composition with the plant, partthereof, or seedling or seed thereof (the “target”) can include anymethod known in the art, including, without limitation, spraying thetarget with the composition, dipping the target into the composition,soaking or submersing the target in the composition, coating the targetwith the composition, or adding the composition to the soil in proximityto the target, whereby the composition comes in contact with thecomposition. Optionally, to facilitate the contacting step, thecomposition is in the form of a liquid, such as an aqueous solution oran oil-based mixture. In such embodiments, the composition may furtherinclude a solubilizing agent that increases the solubility of the COwithin the liquid composition, and/or a surfactant or wetting agent thatfacilitates maximum contact between the liquid composition and the plantor seed surface to which it is applied.

The period for which the target is contacted with the composition canvary. In some embodiments, the composition is contacted with the plant,plant part, seedling or seed for about 5 minutes to about a week.Optionally, the contacting step occurs for a period that falls within arange having a lower value of about 10 minutes, about 20 minutes, about30 minutes, about 40 minutes, about 50 minutes, about one hour, abouttwo hours, about three hours, about four hours, about five hours, aboutsix hours, about seven hours, about eight hours, about nine hours, aboutten hours, about eleven hours, or about twelve hours. Optionally, thecontacting step occurs for a period that falls within a range having anupper value of about 20 minutes, about 30 minutes, about 40 minutes,about 50 minutes, about one hour, about two hours, about three hours,about four hours, about five hours, about six hours, about seven hours,about eight hours, about nine hours, about ten hours, about elevenhours, about twelve hours, about one day, about two days, about threedays, or about four days.

The concentration of the CO in the composition can vary. Optionally, theconcentration of the CO within the composition falls within a rangehaving a lower value of about 10⁻⁴ M, about 10⁻⁵ M, about 10⁻⁶ M, about10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M, orabout 10⁻¹² M. Optionally, the concentration of the CO within thecomposition falls within a range having an upper value of about 10⁻³ M,about 10⁻⁴ M, about 10⁻⁵ M, about 10⁻⁶ M, about 10⁻⁷ M, about 10⁻⁸ M,about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M, or about 10⁻¹² M.

The compositions may include a single type of CO, but may also includemixtures of two or more distinctly different COs, as described above.

C. Examples

The following Examples are offered by way of illustration only, and notby way of limitation.

Example 1 CO4 and CO8 Activate Symbiotic Signalling and Promote RootSystem Development in Rice

Plants establish root symbioses with arbuscular mycorrhizal fungi tofacilitate nutrient acquisition. Establishment of this interactionrequires plant recognition of diffusible signals from the fungus,including lipochitooligosaccharides (LCDs) and chitooligosaccharides(COs). Nitrogen-fixing rhizobia bacteria that form symbioses withleguminous plants also signal to their hosts via LCOs (Nod factors). Inlegumes, it is thought that both the mycorrhizal and rhizobial symbiosesuse a common signaling pathway.

In this example, we have assessed the induction of symbiotic signallingprocesses by the mycorrhizal (Myc)-produced LCOs and COs in rice, amodel non-leguminous mycorrhizal plant. We show that the chitinoligomers CO4 and CO8, but not Myc-LCOs, activate symbiotic calciumoscillations in rice atrichoblasts, although CO4 and Myc-LCOs combinedwere required to induce calcium spiking in root hair cells. In contrast,lateral root emergence was promoted in rice by non-sulphated-LCO(NS-LC), sulphated-LCO (S-LCO), CO4 or CO8, in a DMI3 and POLLUXdependent manner. Our work demonstrates that COs such as CO4 and CO8 canbe used to promote increased root system development in non-leguminousmycorrhizal plants, such as rice.

Introduction

To better understand the mechanisms by which AM fungi signal tonon-leguminous host plants, we assessed the induction of symbioticsignalling in rice by the AM produced LCOs and COs. We show in thisexample that rice appears to respond primarily to COs for activation ofcalcium oscillations, rather than the LCOs that legumes respond to(although rice does respond to LCOs with the promotion of lateral rootemergence). Furthermore, we show for the first time that COs, such asCO4 and CO8, can be used alone or in combination with LCOs to promoteroot system development in non-leguminous plant species, such as rice.We conclude that COs activate different symbiotic signalling processesin non-leguminous plant species, such as rice, from those activated inlegumes.

Materials and Methods

Seed Preparation, Plant Growth Conditions, and Treatment with LCOs andCOs.

Oryza sativa cv Nipponbare wild-type and Tos17 insertion lines in POLLUX(line NC6453) and DMI3 (line 8513) were used for root architectureexperiments (12, 13). Seeds were prepared by sterilizing with 2% bleachfor 20 minutes, followed by 3, 5-minute rinses with sterile water. Thesterile seeds were then imbibed overnight. Seeds were then plated ondamp germination paper in Petri plates under sterile conditions andgerminated in the dark at 25 degrees for 7 days. Germinated rice plantswere then plated on Fahraeus medium on germination paper and grown at 22degrees under constant light. After 5 days the plants were treated with10⁻⁸M LCOs and COs for 24 hours by submersion and re-plated onto FåhræusMedium 1.5% agar plates (0.132 g/L CaCl2, 0.12 g/L MgSO₄.7H₂O, 0.1 g/LKH₂PO₄, 0.075 g/L Na₂HPO₄.2H₂O, 5 mg/L Fe-citrate, and 0.07 mg/L each ofMnCl₂.4H₂O, CuSO₄.5H₂O, ZnCl₂, H₃BO₃, and Na₂MoO₄.2H₂O, adjusted to pH6.5 before autoclaving). As all signals were suspended in 50% ethanol,the appropriate concentration of ethanol in sterile DI water served as acontrol. Root system architecture was assessed after 2 weeks.

Calcium Imaging.

Mycorrhizal induced calcium responses were measured as describedpreviously (14). Transgenic Oryza sativa Nipponbare lines carryingYellow Cameleon 3.6 (YC3.6) FRET-based calcium sensor was used to detectcalcium spiking. YC3.6 was imaged on a Nikon Eclipse Ti invertedmicroscope (Nikon, Japan) equipped with an OptoLED Illuminator (modelOptoLED, Cairn Research Ltd, UK). YC3.6 was excited at a wavelength of455 nm using a royal blue LED and was captured with a charge-coupleddevice (CCD) camera (model RETIGA-SRV, Qimaging, Canada). Emittedfluorescence was separated by an image splitter with a dichroic mirror(model Optosplit II, Cairn Research Ltd, UK) and then passed through afluorescence filter set. Images were collected every 5 seconds with1-second exposure and analyzed using MetaFluor (Molecular Devices,Sunnyvale, Calif., USA).

Mathematical Analysis of Calcium Oscillations.

For Bayesian Spectrum Analysis we computed the most probable periods inthe time series following published procedures (15). Ten traces pertreatment were analyzed. The joint distributions over the period wereused to characterize each group. The plots show binned data to summarizethe key periods. These ten traces per treatment were also analyzed forinterspike intervals. The point of maximum height for each spike wascomputed after detrending of the time series using a moving averagealgorithm. The distances between these maxima gave rise to an interspikedistribution. We used the non-parametric Mann-Whitney U-test, also knownas the Mann-Whitney-Wilcoxon test (16, 17) to test for significantdifferences between the distributions.

Three traces per treatment, with approximately 80 spikes altogether,were analyzed for calcium spike characteristics. The time series had aninterval of 5 seconds between data points. The traces were detrendedusing a moving average algorithm (18). We then characterized the spikesby the time required for each upward and downward phase. This wascomputed by the number of data points it took from the maximum spikeheight to the baseline fluctuation of the trace. The plots show the meanvalue of the upward and downward phases for each treatment, and theassociated standard deviations are indicated by the error bars.

Measurements of Root Architecture Modifications.

Total lateral roots of rice were enumerated manually two weeks afterapplication of 10⁻⁸M COs and LCOs. As COs and LCOs were suspended in 50%ethanol, the control is sterile deionized water containing theappropriate amount of ethanol. Lateral roots were defined as large andfine lateral roots emerging from crown roots, as well as fine lateralroots emerging from large lateral roots. Root system length was measuredstarting from the root-shoot junction to the tip of the longest root.The number of crown roots was assessed manually as those roots emergingfrom the root-shoot junction. Root type characterizations were basedupon the descriptions in Gutjahr et al. (19). Data were assessed fornormality using the Shapiro-Wilk test (α=0.01), and statisticalsignificance was determined using a paired t-test assuming unequalvariances, or a Mann-Whitney-Wilcoxon Test if normality was not observed(α=0.05). All statistical analysis was conducted using the R softwarepackage (20).

Results

Activation of Calcium Oscillations by Myc-LCOs and COs in Rice.

Mycorrhizal fungi associate with a wide range of plant species, and atleast in rice this association is dependent on the common symbiosissignalling pathway (12, 21-23). Hence, non-legumes should be able torecognize the mycorrhizal produced LCOs and/or COs. Two major species ofLCOs have been characterized from exudates of R. irregularis: LCO-IV(C16:0, S or C18:1, S), which we will refer to as sulfated (S)-LCO andLCO-IV (C16:0 or C18:1), which we will refer to as non-sulfated (NS)-LCO(24). For this study, we used S-LCOs and NS-LCOs that were eitherpurified from R. irregularis exudates or were synthesized ingenetically-modified bacteria as previously described (24). To definethe activation of the symbiosis signalling pathway by the AM-producedLCOs and COs, we assessed their ability to activate calciumoscillations, the pathway's earliest measurable event (3).

Calcium responses were assessed using a stably transformed line of Oryzasativa cv Nipponbare carrying YC3.6. AM fungi have been shown topredominantly colonize the large lateral roots (19), and therefore, wefocused on this root type. No calcium responses were observed followingtreatment with any of the LCOs assessed, but strong calcium oscillationswere observed following treatment with 10⁻⁵M CO4 (FIG. 1). To test anarray of different LCO structures, we analyzed calcium spiking inresponse to Nod factor isolations from the broad host range rhizobialspecies, Rhizobium sp. NGR234, as well as Rhizobium tropici, in additionto the Myc-LCOs. Considering that these LCO treatments were performedwith 10⁻⁵M, we are confident that rice does not respond to the Nodfactors or Myc factors tested. Treatments with 10⁻⁸M CO4 still showedcalcium oscillations in rice epidermal cells, but the robustness of theresponse was reduced and the number of responsive cells was also reduced(FIG. 1). Unlike results reported in M. truncatula, rice appears torespond equally well to both CO4 and CO8 with calcium oscillations (FIG.2).

Mycorrhizal LCOs and COs Induce Rice Root Architecture Modification.

Rice has been shown to respond to AM fungi and exudates from the sporesof AM fungi, with changes to root structure, in particular, thepromotion of lateral root outgrowth (19, 25). These responses wereindependent of the common symbiosis signalling pathway. In contrast, wehave observed CO4 and CO8 induction of the common symbiosis signallingpathway in rice as measured by the activation of calcium oscillations.In an attempt to understand these seemingly contradictory results, wetested the promotion of lateral root outgrowth in rice by S-LCO, NS-LCO,CO4 and CO8. This study showed that NS-LCO, S-LCO, CO4 and CO8 allpromoted lateral root and crown root growth in rice (FIG. 3).Interestingly, root system length was only enhanced upon application ofNS-LCOs (FIG. 3). These results imply that COs and LCOs activate twomodalities of signalling in rice: calcium oscillations that areactivated by COs and a separate signalling pathway activated by bothLCOs and COs that is associated with changes to root architecture.

Root Architecture Modification by COs and LCOs are DMI3 and POLLUXDependent.

To assess the role of the common symbiotic pathway in regulating rootarchitecture modifications in response to purified LCOs and COs, wequantified root responses to mycorrhizal signals in rice knock-outmutants of pollux (upstream of calcium spiking) and dmi3 (downstream ofcalcium spiking). We found that lateral root growth promotion by LCOsand CO4 was dependent on both POLLUX and DMI3, while the response to CO8was dependent on POLLUX (FIG. 4). The increase in crown root growth byboth LCOs and COs was dependent upon DMI3 and POLLUX, and there weresignificantly fewer crown roots in response to all treatments in thepollux mutant (FIG. 4). Finally, the overall root length increaseobserved in response to NS-LCO was dependent upon both POLLUX and DMI3(FIG. 4). These results demonstrate that root architecture changes inresponse to purified mycorrhizal signals require proteins of the commonsymbiotic pathway.

Both LCOs and COs are Required to Induce Calcium Spiking inTrichoblasts.

Rhizobia colonize legumes by root hair cells (trichoblasts), whereas AMfungi colonize roots via non-root hair epidermal cells (atrichoblasts).Thus, these different root epidermal cell types may respond differentlyto COs and LCOs. To test this, we directly compared trichoblast andatrichoblast responses using high concentrations of CO4 in rice. Calciumoscillations observed in rice following treatments of CO4 wererestricted to atrichoblasts, with no responses in trichoblasts even withCO4 treatments of 10⁻⁵ M (FIG. 5). This preferential nature of riceatrichoblasts to respond to the AM signals is consistent with apreference for AM fungi to colonize the root via atrichoblasts (26). Itis possible that either AM fungi produce signalling molecules other thanS-LCO, NS-LCO and CO4 that induce calcium oscillations in ricetrichoblasts, or that the mix of signalling molecules is important. Totest this, we assessed induction of calcium oscillations by an equimolarmix of 10⁻⁵ M S-LCO, NS-LCO, and CO4. Strikingly, we observed calciumspiking in rice root hair cells when treated with this mix of signalmolecules, yet these signal molecules, when applied individually at 10-5M, did not induce calcium spiking (FIG. 6).

Discussion

COs and LCOs Act Synergistically as Symbiotic Signals in Rice.

AM fungi signal to the host plant via diffusible signals (14, 25-27),and at least some of these signals are LCOs (24) and COs (9). In thisexample, we show that the AM-produced COs can activate calciumoscillations in rice. S-LCO and NS-LCO were purified from exudates of AMfungi based on their capability to activate symbiotic responses in M.truncatula that were dependent on the common symbiosis signallingpathway (24). The fact that these LCOs do not trigger calcium spiking inrice may reflect this selectivity in their initial identification.However, rice can sense LCOs since the mix of Myc-LCOs and CO4 activatedcalcium oscillations in rice root hair cells and LCOs can also promotelateral and crown root growth. Therefore, the absence of calciumresponses in rice to the LCO treatments alone does not indicate a lackof response of LCOs by rice.

Our work has revealed a close correlation between the cell-type and itsresponsiveness to LCOs and CO4. We observed that calcium responses toCOs were restricted to atrichoblasts in rice. This preferential responsein atrichoblasts correlates well with a preferential colonization ofatrichoblasts by AM fungi (26). However, we observed calciumoscillations in rice trichoblasts in treatments where LCOs and CO4 werecombined. It would appear that responses in rice trichoblasts are atleast partially explained by the mix of LCOs and CO4 produced by the AMfungus. Interestingly, it was shown some years ago that a mix of Nodfactors and COs was better at inducing nodulation associated geneexpression in soybean than Nod factor treatments alone (28). Perhapsthese earlier observations reflect responses to AM fungi, rather thanwhat was previously thought to be a rhizobial response. Alternatively, amix of LCOs and COs may be relevant in rhizobial interactions as well asAM associations.

Multiple Pathways Mediate LCO and CO Responses in Rice.

We established that rice can distinguish between LCOs, CO4, and CO8, andresponds accordingly with either calcium oscillations and/or rootarchitecture modifications. The fact that rice responds to AM fungi withat least two separate signalling pathways has already been shown (23),and the promotion of lateral roots by AM fungi in a manner independentof the common symbiosis signalling pathway was also already shown (25).Thus, there is ample evidence in rice for two pathways of symbiosissignalling. Our work shows that root architecture modification inresponse to LCOs and CO4 requires the common symbiotic pathway; however,CO8 does not require DMI3. The ability of AM fungi to stimulate lateralroot emergence independent of the symbiotic pathway may indicate thatthe plant responds differently to a mixture of signals and stimuli thanit does to purified signals and that during symbiosis the pathwaygoverning root architecture modification does not require calciumspiking to be initiated.

In Arabidopsis, lateral root development is under the control of auxinsignalling modules. Under high auxin conditions, lateral root foundercells polarize and divide (29). Further rounds of cell division resultin lateral root emergence at specific sites in the root. The processleading to lateral root emergence is similar in rice and using theDR5:GUS auxin reporter system, auxin was shown to accumulate in emerginglateral roots (30). Under high auxin concentrations, AUX/IAA proteinsare degraded. AUX/IAA proteins repress ARF transcriptional activators,and thus their degradation leads to the transcription ofauxin-responsive genes (29). Auxin positively regulates lateral rootformation, as a rice plant containing a constitutively active version ofIAA13 has fewer lateral roots than wild type (31). Interestingly, auxinsignalling is also implicated in the production of crown roots in rice(32, 33). It seems likely, therefore, that the application of LCOs andCOs activates the auxin-dependent lateral root and crown root emergenceprograms. Given that this phenotype was dependent on DMI3 and POLLUX inthe case of Myc-LCOs and CO4, it may be that there is cross talk betweenthe common symbiosis pathway and auxin signaling, which results inincreased lateral root emergence and crown root growth. Assessingexpression of auxin-responsive genes in Ospollux and Osdmi3 mutants inresponse to COs and LCOs may reveal the mechanisms of this signallingpathway.

CONCLUSION

AM fungi have the distinctive capability of colonizing a broad group ofplants. In this example, we demonstrate that CO4 and CO8 form at leastpart of the spectrum of AM symbiotic signals that can be recognized by avariety of plant species to activate a range of symbiotic signallingprocesses. More specifically, both CO4 and CO8 can be used to promoteincreased root system development in non-leguminous plants, such asrice.

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Example 2 CO4 does not Promote Root System Development in Legumes

This example shows that the results obtained in Example 1 using rice, anon-leguminous mycorrhizal plant model, cannot be replicated in legumes.Specifically, we demonstrate that in M. truncatula, stimulation oflateral root emergence occurred following treatment with Myc-LCOs, butnot following treatment with CO4. In contrast, Example 1 showed thatboth Myc-LCOs and CO4 (along with CO8) promoted increased root systemdevelopment in rice. This work indicates that legumes and non-legumesdiffer in their perception of Myc-LCO and CO signals, suggesting thatlegume and non-legume species respond to different components in the mixof signals produced by arbuscular mycorrhizal fungi.

Results

LCOs, CO4, and CO8 Promote Root Development in Rice, while LCOs, but notCOs, Promote Root Development in the Legume M. truncatula.

We tested the promotion of lateral root outgrowth in rice by S-LCO,NS-LCO, CO4, and CO8, and found that all four molecules could promotelateral root outgrowth and enhance overall root system growth (seeExample 1). In contrast to what we observed in rice, using the protocolof Example 1, CO4 could not induce lateral root emergence in M.truncatula (FIG. 7), but the Myc-LCOs can activate this response(Maillet, F., et al. (2011), Nature 469: 58-63). This demonstrates thatlegumes and non-legumes differ fundamentally in their response to thespectrum of Myc-LCOs and COs for modifications to root architecture.Accordingly, the disclosed method is limited in scope to treatingnon-leguminous plants, or seeds, seedlings or plant parts thereof.

Example 3 The Effect of CO4 and CO8 Seed Treatment on Growth andDevelopment of Hard Red Spring Wheat, Rice and Corn

This example demonstrates that CO4 and/or CO8 when applied to the seedsof three different grain crop plants, promotes the growth of the plants,as measured by one or more parameters of plant growth or development.

Experiment 1 Effects of CO4 and CO8 Treatment on Hard Red Spring Wheat(HRSW) In Vitro

Methods

Hard red spring wheat seeds were surface sterilized using ethanol andbleach. The sterilized seeds were then divided among 4 treatment groupsof 5 grams each into 50 ml falcon tubes. To the control tube, 0.5%ethanol in water was added. To the CO4 tube, 125 μl of 10⁻⁶ M CO4solution was added. To the CO8 tube, 125 μl of 10⁻⁶ M CO8 solution wasadded. To the “Consensus” tube, 125 μl of 10⁻⁶ M solution of chitosan(CONSENSUS® chitosan; Loveland Products, Inc., Loveland, Colo.) wasadded. Each tube was then shaken to coat the seeds. The coated seedswere then placed on sterile Petri dishes in a hood and were left to dryovernight in the hood.

The seeds were then germinated on damp germination paper in sterilePetri dishes for 4 days. The resulting seedlings were transferred toFahräeus medium plates containing damp germination paper, and the plateswere wrapped in Parafilm®. Fahräeus medium contains 0.5 m M MgSO₄, 0.7mM KH₂PO₄, 0.4 mM Na₂HPO₄, 0.02 mM Fe-EDTA, 0.01 mM MnSO₄, 0.007 mMCuSO₄, 0.006 mM ZnSO₄, 0.016 mM H₃BO₃, 0.001 mM Na₂MoO₄, and 15 g/L Agarfor plant tissue, adjusted to pH 6.5 before autoclaving. The plants weregrown at room temperature under continuous light for 4-5 days. Thenumber of primary roots (PR), lateral roots (LR), root system length(RSL) and dry weight (DW) was then measured for all plants.

Results

The results are tabulated in Table 1 below:

TABLE 1 Measured Parameters for Control, CO8, CO4 and ConsensusTreatment Groups (Wheat) Number of Number of Primary Lateral Root SystemDry Weight Treatment Roots Roots Length (cm) (g) Control 5.64 3.21 11.820.0194 CO8 5.20 3.00 11.51 0.0191 CO4 5.20 3.73 13.21* 0.0206 Consensus5.27 3.53 11.52 0.0213 *Significant difference at P < 0.05

CO4 treatment significantly increased root system length over thecontrol (see also FIG. 8), indicating that the treatment facilitates atleast one growth parameter in wheat.

Experiment 2 Effects of CO4 and CO8 Treatment on Hard Red Spring Wheatin Pots

Methods

Five grams of hard red spring wheat seeds were added to three separate50 ml tubes. A water solution containing 0.5% ethanol was added to thecontrol tube, 125 μl of 10⁻⁶ M CO4 solution was added to the CO4 tube,and 125 μl of 10⁻⁶ M CO8 solution was added to the CO8 tube. Each tubewas then shaken to coat the seeds. Seeds were potted immediatelyfollowing treatment into moistened SUNGRO® potting mix in 4×6×6 cm pots.Plants were watered by pouring water into the tray containing the potsup to about ¾ inch high.

After sowing the seeds, the pots were randomly distributed (16 pots pertreatment) throughout the tray and placed underneath continuous light atroom temperature for 1 week. Liquid Fahräeus medium (plant fertilizersolution; see above) was applied every 2 days or when necessary. After aweek, the pots were placed underneath 10 h light and 14 h dark cycle atroom temperature for 1 week. After 1 week underneath 10 h light and 14 hdark cycle (2 weeks after planting), the plants were carefully removedfrom the pots, and as much soil as possible was shaken off. Theremaining soil was removed by submerging the root system into a beakerof water.

The number of primary roots (PR), lateral roots (LR), root system length(RSL) and dry weight (DW) was then measured for all plants.

Results

The results are tabulated in Table 2 below:

TABLE 2 Measured Parameters for Control, CO8 and CO4 Treatment Groups(Wheat) Number of Number of Primary Lateral Root System Dry WeightTreatment Roots Roots Length (cm) (mg) Control 3.30 14.45 10.70 24.03CO8 3.35 14.30 12.22 32.38* CO4 3.15 16.25 11.75 30.20* *Significantdifference at P < 0.05

Both CO4 and CO8 seed treatments significantly increased dry weight (ameasurement of total plant growth) in the wheat plants (see also FIG.9), confirming that such treatments facilitate plant growth in wheat. Asimilar dry weight increase was not shown with plants grown in plates inExperiment 1 above, likely because of the limitations on growth thatplates impose. Average root system length was also higher for both CO4and CO8 treatments, although the increased length was not great enoughto be significant at P<0.05.

Together, the results of Experiment 1 and 2 demonstrate that CO4 and/orCO8 treatment of wheat seeds facilitates growth and development of thewheat plants that germinate from the treated seeds.

Experiment 3 Effects of CO4 and CO8 Treatment on Rice In Vitro

Methods

Rice seeds were sterilized using 2% bleach. The sterilized seeds werethen divided among 3 treatment groups of 10 seeds each into 15 ml falcontubes. To the control tube, 0.5% ethanol in water was added. To the CO4tube, 100 μl of 10⁻⁶ M CO4 solution was added. To the CO8 tube, 100 μlof 10⁻⁶ M CO8 solution was added. Each tube was then shaken to coat theseeds. The coated seeds were then placed on sterile Petri dishes in ahood and were left to dry overnight in the hood.

The seeds were then germinated on damp germination paper in sterilePetri dishes for 5 days. The resulting seedlings were transferred toFahräeus medium plates containing damp germination paper, and the plateswere wrapped in Parafilm®. The roots part of the plate was covered withaluminum foil. The plants were grown at room temperature undercontinuous light for 33 days. The length of the shoot and root systemswas measured for all plants.

Results

CO4 treatment significantly increased both shoot and root system lengthcompared with the ethanol control (see FIG. 10). This data supplementsthe data disclosed in Example 1 above indicating that both CO4 and CO8treatment can be used to facilitate growth and development in rice.

Experiment 4 Effects of CO4 and CO8 Treatment on Corn in Pots

Methods

Five grams of corn (maize) seeds were added to three separate 50 mltubes. A water solution containing 0.5% ethanol was added to the controltube, 125 μl of 10⁻⁶ M CO4 solution was added to the CO4 tube, and 125μl of 10⁻⁶ M CO8 solution was added to the CO8 tube. Each tube was thenshaken to coat the seeds. Seeds were potted immediately followingtreatment into moistened SUNGRO® potting mix in 4×6×6 cm pots. Plantswere watered by pouring water into the tray containing the pots up toabout ¾ inch high.

After sowing the seeds, the pots were randomly distributed (16 pots pertreatment) throughout the tray and placed underneath continuous light atroom temperature for 1 week. Liquid Fahräeus medium (plant fertilizersolution; see above) was applied every 2 days or when necessary. After aweek, the pots were placed underneath 10 h light and 14 h dark cycle atroom temperature for 1 week. After 1 week underneath 10 h light and 14 hdark cycle (2 weeks after planting), the plants were carefully removedfrom the pots, and as much soil as possible was shaken off. Theremaining soil was removed by submerging the root system into a beakerof water.

The number of primary roots (PR), lateral roots (LR), root system length(RSL) and dry weight (DW) was then measured for all plants.

Results

The results are tabulated in Table 3 below. As shown in Table 3 and FIG.11, corn plants resulting from seeds receiving both CO4 and CO8treatments exhibited increased number of lateral roots, as compared tocorn plants resulting from seeds receiving the control treatment.However, the increased lateral root development with CO8 treatment wasnot great enough to be significant at P<0.1, likely due to highvariability. CO4 did significantly increase lateral root growth atP<0.1. This experiment demonstrates that the disclosed seed treatmentmethod can be used to facilitate growth in corn.

TABLE 3 Measured Parameters for Control, CO8 and CO4 and TreatmentGroups (Corn) Average Root Dry Treatment Seminal Roots Lateral RootsSystem Length Weight Control 4.40 38.95 21.53 0.1176 CO4 4.45 45.00*23.76 0.1225 CO8 4.75 44.95 21.42 0.1229 *Significant difference at P <0.1

Example 4 The Effect of CO4 and CO8 Foliar Treatment on Root/ShootGrowth of Rice

This example extends the results of the previous examples to foliartreatment in rice. Specifically, foliar treatment with CO4 in rice isshown to facilitate both shoot growth and root growth.

Methods

Rice seeds were surface sterilized using 2% bleach. Seeds were thengerminated on damp germination paper in sterile Petri dishes for 5 days.The seedlings were then transferred to Fahraeus medium plates containingdamp germination paper. The plates were wrapped in Parafilm®, and theroots part of the plate covered with aluminum foil.

The plants were grown at room temperature under continuous light for 21days. The plants were then transferred to moistened SUNGRO® soil pots(8×8×10 cm) and grown for 3 days under greenhouse condition. The plantswere hydrated by pouring water into the tray underneath the pots.

The soil surface was then covered with plastic wrap to prevent anyliquid droplets from contacting the soil. Using painting brushes, thefront, and back of the plant leaves were treated with 3 ml of chitinderived compounds (CO4 and CO8) at 10⁻⁶ M or a control (0.5% ethanol),each including 0.05% of Silwet L-77 (surfactant). Eleven plants weretreated with each solution. The plants were subsequently grown for 14days, and the shoot length was measured. Once a week, half-strengthHoagland solution was added into the trays underneath the pots. Theplants were grown for another 4 days, and the lengths of the shoot androot systems were measured.

Results

CO4 foliar treatment on rice significantly increased shoot length at day14 after treatment, and the significance was even higher at day 18 (FIG.12A). CO4 treatment also significantly promoted root system length atday 18 (FIG. 12B). In sum, this example shows that foliar application ofCO4 in rice promotes plant growth in the rice.

Example 5 CO4 and CO8 Seed Treatment Reduce Root Growth in an ExemplaryLegume

Consistent with the results reported in Example 2, this example confirmsthat the results reported in various non-leguminous plants (see Examples1, 3 and 4) cannot be replicated in legumes. Thus, the disclosed methodis limited to non-leguminous plants.

Methods

Five grams of pea seeds (the legume Pisum sativum) were added to threeseparate 50 ml tubes. A water solution containing 0.5% ethanol was addedto the control tube, 125 μl of 10⁻⁶M CO4 solution was added to the CO4tube, and 125 μl of 10⁻⁶ M CO8 solution was added to the CO8 tube. Eachtube was then shaken to coat the seeds. Seeds were potted immediatelyfollowing treatment into moistened SUNGRO® potting mix in 4×6×6 cm pots.Plants were watered by pouring water into the tray containing the potsup to about ¾ inch high.

After sowing the seeds, the pots were randomly distributed (16 pots pertreatment) throughout the tray and placed underneath continuous light atroom temperature for 1 week. Liquid Fahräeus medium (plant fertilizersolution; see above) was applied every 2 days or when necessary. After aweek, the pots were placed underneath 10 h light and 14 h dark cycle atroom temperature for 1 week. After 1 week underneath 10 h light and 14 hdark cycle (2 weeks after planting), the plants were carefully removedfrom the pots, and as much soil as possible was shaken off. Theremaining soil was removed by submerging the root system into a beakerof water.

The number of primary roots (PR), lateral roots (LR), root system length(RSL) and dry weight (DW) were then measured for all plants.

Results

The results are tabulated in Table 4 below:

TABLE 4 Measured Parameters for Control, CO8 and CO4 Treatment Groups(Peas) Average Lateral Tap Root Treatment Primary Roots Roots Length DryWeight Control 18.45 22.45 10.31 .15 CO4 14.20* 13.35* 10.25 .13 CO415.50* 15.00* 10.28 .14 *Significant difference at P < 0.05

In contrast to the results demonstrated with non-leguminous plants, CO4,and CO8 seed treatments significantly decreased the number of primaryand lateral roots of pea seedlings compared to the control. None of theother variables were affected by the CO treatment.

Together, the results of Example 2 and this example demonstrate that thedisclosed chitin oligomers cannot be used to promote the growth and/ordevelopment of leguminous plants. Accordingly, the disclosed method islimited to promoting the growth and/or development of non-leguminousplants.

The examples and embodiments described herein are for illustrativepurposes only. Various modifications or changes in light thereof will besuggested to persons skilled in the art and are to be included withinthe spirit and purview of this application and scope of the appendedclaims. All publications, patents, and patent applications cited hereinare hereby incorporated by reference in their entirety for all purposes.

We claim:
 1. A method for stimulating the growth of a non-leguminousplant, comprising contacting a non-leguminous plant, a part thereof, ora seedling or seed thereof with a composition comprising achitooligosaccharide (CO) having the formula:

wherein n is 0, 1, 2, 3, 4, 5, or 6; R1 is selected from the groupconsisting of —H, —CH₃, —COCH₃, —SO₃H, —SO₃Na, arabinose, methylatedarabinose, fucose, and methylated fucose; R2 is selected from the groupconsisting of —H, —CH₃, —COCH₃, —SO₃H, —SO₃Na, arabinose, methylatedarabinose, fucose, and methylated fucose; and each R3 is independentlyselected from the group consisting of —H and —COCH₃; whereby the growthof the plant is stimulated.
 2. The method of claim 1, wherein thecomposition is contacted with one or more leaf or root surfaces of thenon-leguminous plant.
 3. The method of claim 2, wherein the compositionfurther comprises a surfactant.
 4. The method of claim 1, wherein thecomposition is contacted with a seedling, seedling part or seed of thenon-leguminous plant.
 5. The method of claim 4, wherein the seedlingpart or seed of the non-leguminous plant is submerged in andsubsequently removed from the composition.
 6. The method of claim 1,wherein the composition is contacted with the plant, plant part,seedling or seed for about 1 hour to about 96 hours.
 7. The method ofclaim 6, wherein the composition is contacted with the plant, plantpart, seedling or seed for about 6 hours to about 48 hours.
 8. Themethod of claim 1, wherein the concentration of the CO in thecomposition is within the range of about 10⁻³M to about 10⁻¹⁰ M.
 9. Themethod of claim 8, wherein the concentration of the CO in thecomposition is within the range of about 10⁻³M to about 10⁻⁹M.
 10. Themethod of claim 9, wherein the concentration of the CO in thecomposition is within the range of about 10⁻³M to about 10⁻⁸M.
 11. Themethod of claim 1, wherein the composition further comprises water andalcohol.
 12. The method of claim 11, wherein the alcohol is ethanol. 13.The method of any of claim 1, wherein the non-leguminous plant is amonocotyledon.
 14. The method of claim 12, wherein the monocotyledon isa cereal grain.
 15. The method of claim 13, wherein the cereal grain isselected from the group consisting of rice, wheat, and corn.
 16. Themethod of any of claim 1, wherein R1 is —H, R2 is —H, and each R3 is—COCH₃.
 17. The method of claim 16, wherein n is 2 (the compound istetra-acetyl-chitotetraose, CO4) or 6 (the compound isocta-N-acetyl-chitooctaose, CO8).
 18. The method of claim 17, wherein nis 2 (the compound is tetra-N-acetyl-chitotetraose, CO4).
 19. The methodof claim 17, wherein n is 6 (the compound istetra-N-acetyl-chitotetraose, CO4).
 20. The method of claim 1, whereinthe growth of the non-leguminous plant that is stimulated is selectedfrom the group consisting of increased number of crown roots, increasednumber of lateral roots, increased root length, increased dry weight,increased shoot length, and combinations thereof, as compared to thesame quantity measured in the same type of non-leguminous plant whereinthe plant, part thereof, or seedling or seed thereof has not beencontacted with the composition.